Abstract
Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins. Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. We have cloned and characterized a novel 190-kDa member of this family from a rat kidney cDNA library, which we term AnkG190 based on the predicted size and homology with the larger neuronal AnkG isoform. AnkG190 displays a unique 31-residue amino terminus, a repeats domain consisting of 24 repetitive 33-residue motifs, a spectrin binding domain, and a truncated regulatory domain. Probes derived from the unique amino terminus hybridize to an 8-kilobase message exclusively in kidney and lung and specifically to the kidney outer medullary collecting ducts by in situ hybridization. Transfections of Madin-Darby canine kidney and COS-7 epithelial cell lines with a full-length AnkG190 construct result in (a) expression at the lateral plasma membrane, (b) functional assembly with the cytoskeleton, and (c) interaction with at least one membrane protein, the Na,K-ATPase. Two independent Na,K-ATPase binding domains on AnkG190 are demonstrated as follows: one within the distal 12 ankyrin repeats, and a second site within the spectrin binding domain. Thus, ankyrins may interact with integral membrane proteins in a pleiotropic manner that may involve complex tertiary structural determinants.
Highlights
Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins
Ankyrins are a family of conserved proteins that have emerged as crucial adapter molecules mediating such linkages, since they possess binding sites for several integral membrane proteins as well as for cytoskeletal elements (4 – 6)
Sequence searches with GenBankTM reveal that rat AnkG190 is only 80% homologous to mouse epithelial ankyrin (Ank3), with significant sequence diverge in the unique amino-terminal residues and within the regulatory domain, where rat AnkG190 is completely devoid of a 197-residue motif present in mouse Ank3 [12]
Summary
Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. Spliced isoforms of this parent AnkG protein include a 215-kDa peptide encoded for by a 7-kb mRNA in mouse epithelial tissues [12], a Golgi-associated 116-kDa species (AnkG119) expressed predominantly in epithelial cells and muscle [13], and small 120/100-kDa Ank molecules associated with mouse macrophage lysosomes [14]. Additional immunoreactive Ank isoforms at 200, 170, 120, and 105 kDa have been detected in mouse tissues, but incompletely characterized [12] Such rich isoform diversity may be especially critical to the maintenance of a specific pattern of membrane protein distribution in polarized epithelial cells such as those lining various segments of the kidney tubules, each of which vectorially transports specific ions and nutrients. The polarized distribution of ankyrin (and Na,K-ATPase) is markedly disrupted following an ischemic kidney injury and is restored following recovery [20, 21], suggesting an important role for the 190-kDa ankyrin in the generation and maintenance of renal cell polarity
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